Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression

Lindqvist, Arne; Zon, Wouter van; Rosenthal, Christina Karlsson; Wolthuis, Rob M. F.

doi:10.1371/journal.pbio.0050123

Cited by 155 publications

(186 citation statements)

References 54 publications

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“…Using immunofluorescence and subcellular compartment fractionation followed by immunoblotting, we detected that hRFPL1 was mostly localized in the cell cytoplasm (Figure 5a and b), similar to cyclin B1 and Cdc2 subcellular localization during S and G 2 phases. 15 Thus, we hypothesized that hRFPL1 could alter cyclin B1 and Cdc2 accumulations during this period. To address this question, we synchronized HeLa cells in G 1 phase (cell population going from 47-18-35 to 71-18-11% after mimosine treatment, in G 1 , S and G 2 -M phase, respectively) and examined by immunoblotting cyclin B1 and Cdc2 levels over time following release from cell-cycle block.…”

Section: Resultsmentioning

confidence: 99%

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation

et al. 2010

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Ret finger protein-like 1 (RFPL1) is a primate-specific target gene of Pax6, a key transcription factor for pancreas, eye and neocortex development. However, its cellular activity remains elusive. In this article, we report that Pax6-elicited expression of the human (h)RFPL1 gene in HeLa cells can be enhanced by in vivo p53 binding to its promoter and therefore investigated the hypothesis that hRFPL1 regulates cell-cycle progression. Upon expression in these cells, hRFPL1 decreased cell number through a kinase-dependent mechanism as PKC activates and Cdc2 inhibits hRFPL1 activity. hRFPL1 antiproliferative activity led to an increased cell population in G 2 /M phase and specific cyclin B1 and Cdc2 downregulations, which were precluded by a proteasome inhibitor. Specifically, cytoplasm-localized hRFPL1 prevented cyclin B1 and Cdc2 accumulation during interphase. Consequently, cells showed a delayed entry into mitosis and cell-cycle lengthening resulting from a threefold increase in G 2 phase duration. Given previous reports that RFPL1 is expressed during cell differentiation, its impact on cell-cycle lengthening therefore provides novel insights into primate-specific development. The human Ret finger protein-like (hRFPL)1,2,3 genes (OMIM 605968, 605969, 605970) are recently identified targets of Pax6, which is notably a key transcription factor for pancreas, eye and neocortex development. [1][2][3] In this line, high hRFPL1,2,3 expression is found at the onset of neurogenesis in differentiating embryonic stem cells and in the developing neocortex. 4 In addition, the study of RFPL1,2,3 evolutionary history revealed that they are only found in Old World monkeys and great apes and show features believed to be important for human brain evolution. 4 Yet, the cellular activity of RFPL1,2,3 is still unknown. A murine RFPL (mRFPL) protein, encoded by an ancestral gene not belonging to the RFPL1,2,3 gene subfamily, 4 has also been cloned. 5 Previously reported as being expressed only in testis, ovaries and oocytes, 5,6 mRFPL has been shown to interact with the destruction box motif of cyclin B1 -the Cdc2 activating partner for driving germ cells through metaphases I and II 7 -and with proteins of the proteasome, speculating on mRFPL ability to elicit cyclin B1 degradation to control meiosis progression. 6 However, the fact that cyclin B1 and Cdc2 also form a key complex for controlling cell entry into mitosis 8 and our recent observations that the RFPL genes are also expressed in tissues in which cells divide mitotically 4 suggest that the RFPL proteins could regulate other aspects of cell division.We therefore examined RFPL-mediated control of mitotic cell-cycle progression by focusing on hRFPL1. Because no endogenously hRFPL1-expressing cell type suitable for this kind of study has been reported to date, we examined the influence of hRFPL1 gain of function on HeLa cells, a reference cell system for examining cell-cycle regulation. We report that hRFPL1 is an antiproliferative gene that controls G 2 -M phase transition, ...

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Section: Resultsmentioning

confidence: 99%

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation

et al. 2010

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“…Immunofluoresence was performed as described in the study by Lindqvist et al (2007). Images were acquired on a Zeiss LSM510 META microscope (Zeiss, Sliedrecht, The Netherlands).…”

Section: Immunofluorescencementioning

confidence: 99%

Oncogenic KRAS sensitises colorectal tumour cells to chemotherapy by p53-dependent induction of Noxa

et al. 2010

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BACKGROUND: Oxaliplatin and 5-fluorouracil (5-FU) currently form the backbone of conservative treatment in patients with metastatic colorectal cancer. Tumour responses to these agents are highly variable, but the underlying mechanisms are poorly understood. Our previous results have indicated that oncogenic KRAS in colorectal tumour cells sensitises these cells to chemotherapy. METHODS: FACS analysis was used to determine cell-cycle distribution and the percentage of apoptotic and mitotic cells. A multiplexed RT -PCR assay was used to identify KRAS-controlled apoptosis regulators after exposure to 5-FU or oxaliplatin. Lentiviral expression of short-hairpin RNAs was used to suppress p53 or Noxa. RESULTS: Oncogenic KRAS sensitised colorectal tumour cells to oxaliplatin and 5-FU in a p53-dependent manner and promoted p53 phosphorylation at Ser37 and Ser392, without affecting p53 stabilisation, p21 induction, or cell-cycle arrest. Chemotherapy-induced expression of the p53 target gene Noxa was selectively enhanced by oncogenic KRAS. Suppression of Noxa did not affect p21 induction or cell-cycle arrest, but reduced KRAS/p53-dependent apoptosis after exposure to chemotherapy in vitro and in tumour xenografts. Noxa suppression did not affect tumour growth per se, but strongly reduced the response of these tumours to chemotherapy. CONCLUSION: Oncogenic KRAS determines the cellular response to p53 activation by oxaliplatin or 5-FU, by facilitating apoptosis induction through Noxa.

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“…Several studies have indicated that Cdk1-Cyclin B regulates microtubule dynamics (Verde et al, 1990;Fourest-Lieuvin et al, 2006). It is proposed that the gradual increase in Cdk1-Cyclin B activity prepares the cell to exit mitosis and the absence of high Cdk1-Cyclin B activity levels are thought to cause a delay in mitosis (Lindqvist et al, 2007).…”

Section: Figure 13 Cdk1 Activation Pathwaymentioning

confidence: 99%

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

Smith¹,

Dejsuphong

Balestrini³

et al. 2009

Nat Cell Biol

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The effects of ATM and ATR signalling induced by chromosomal breakage have been described extensively in modulating cell cycle progression up to the onset of mitosis.However, DNA damage checkpoint responses in mitotic cells are not well understood.This thesis reports on the effects of double strand breaks on the progression of mitosis.We found ATM and ATR activation can occur in mitotic Xenopus laevis egg extract and the induction of ATM and ATR by chromosomal breakages inhibits spindle assembly in both Xenopus egg extract and somatic cells. The delay in mitotic progression induced by ATM and ATR was found not to involve major spindle assembly factors activities such as, Cdk1, Plx1 and RCC1/Ran-GTP. However, normal anastral spindles formation around linear DNA coated beads, which can activate ATM and ATR, linked centrosome-driven spindle assembly to ATM and ATR dependent spindle defects. cDNA expression library screening was undertaken to identify novel ATM and ATR targets in this mitotic checkpoint pathway, through which the novel centrosomal protein XCEP63 was identified as a likely candidate. Data obtained from depletion and reconstitution of XCEP63 in Xenopus egg extract established that normal centrosome-driven spindle assembly requires XCEP63. Moreover, ATM and ATR phosphorylates XCEP63 on serine 560 and promotes delocalisation from the centrosome. ATM and ATR inhibition or addition of non-phosphorylable XCEP63 recombinant protein mutated at serine 560 prevents spindle assembly abnormalities.These findings suggest that ATM and ATR regulate mitotic events by targeting XCEP63 and centrosome-dependent spindle assembly. This pathway may provide support for DNA repair processes or regulate cell survival in the presence of mitotic DNA damage. 3

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Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression

Cited by 155 publications

References 54 publications

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation

Oncogenic KRAS sensitises colorectal tumour cells to chemotherapy by p53-dependent induction of Noxa

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

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